Figure 3

NMDAR-dependent accumulation of synaptic vesicle proteins at developing presynaptic terminals is independent of postsynaptic NMDARs and bi-directional. (A) Synapsin accumulates at presynaptic terminals induced by contact with neuroligin-expressing HEK-293 cells. The neuroligin-expressing cell is identified by co-expression of actin-GFP (green) and is outlined in both panels (white). Axons were labeled with tau-1 antibodies to identify contacts and are visible in the left panel (green, highlighted with yellow dashed lines). Induced synapsin puncta are indicated by arrows (right panel). The left panel also shows the overlay of the synapsin (red) and PSD95 (blue). PSD95 label does not co-localize with induced synapsin puncta. Scale bar: 10 μm. (B) Accumulation of synapsin is decreased by APV at presynaptic terminals that lack postsynaptic partners. (C) Direct activation of NMDARs via treatment with NMDA (0.015 mM for 24 h) increased the fluorescence intensity of synapsin puncta (P < 0.0001). Data are normalized to the control mean and presented as normalized mean ± standard error of the mean. The numbers of puncta quantified for each condition are indicated on the plots in (B, C). *, significant difference (P-values are indicated in the figure).